CN111603474A

CN111603474A - Application of glaucescent fissistigma root extract C21 steroid in preparation of medicine for promoting gastric cancer cell apoptosis

Info

Publication number: CN111603474A
Application number: CN202010117125.8A
Authority: CN
Inventors: 张威; 王震; 应优敏; 李凯强; 张昱; 陈以栗; 郝珂
Original assignee: Zhejiang Provincial Peoples Hospital
Current assignee: Zhejiang Provincial Peoples Hospital
Priority date: 2020-02-25
Filing date: 2020-02-25
Publication date: 2020-09-01

Abstract

The invention discloses application of a C21 steroid in preparation of a medicine for promoting cancer cell apoptosis, wherein the C21 steroid inhibits cell proliferation by inhibiting autophagy to promote cell apoptosis. The C21 steroid is a glaucescent fissistigma root extract, and the C21 steroid inhibits gastric cancer cell proliferation by inhibiting autophagy and promoting apoptosis. The C21 steroid disclosed by the invention can obviously inhibit the growth of cancer cells and promote the occurrence of apoptosis, and provides a theoretical basis for disclosing a new mechanism of the anti-gastric cancer activity of the C21 steroid.

Description

Application of glaucescent fissistigma root extract C21 steroid in preparation of medicine for promoting gastric cancer cell apoptosis

Technical Field

The invention relates to the technical field of biology, in particular to application of a glaucescent fissistigma root extract C21 steroid in preparation of a medicine for promoting gastric cancer cell apoptosis.

Background

Gastric cancer is the third most common cancer worldwide and is one of the leading causes of cancer-related deaths. The lack of effective treatment results in short life cycle and poor quality of life for cancer patients. At present, gastric cancer patients are treated mainly by surgical resection and chemotherapy, but the treatment effect is limited. Therefore, there is an urgent need to develop new treatment regimens to improve patient prognosis.

Autophagy is a conserved eukaryotic catabolic reaction that catabolizes cytoplasmic proteins and organelles. As a cytoprotective process, it helps maintain the homeostasis of cancer cells and serves as an alternative energy source in harsh environments. This suggests that autophagy may have a protective role in the development of tumors. Recent studies have shown that key autophagy genes, including autophagy-related genes (ATGs), can cause cellular ROS damage through cell death. Cully et. found that modulating the expression of the cancer suppressor genes PTEN and p53 could positively modulate autophagy. It is well known that oncogene products such as BCL2 anti-apoptotic proteins interact with evolutionarily conserved autophagy proteins, and that binding to Beclin-1 may help maintain autophagy at a level compatible with cell survival. With respect to gastric cancer, recent studies have shown that autophagy has a dual effect on the disease. Many studies have shown that the anticancer drug akebia saponin PA promotes apoptosis of AGS cells by activating autophagy and apoptosis. In contrast, another study reported that the use of an autophagy inhibitor enhanced the cytotoxicity of an anticancer drug in the gastric cancer cell line SGC-7901. Thus, autophagy may be a potential therapeutic target for the treatment of gastric cancer.

Marsdenia Tenacissima (Marsdenia Tenacissima) is a Fissistigma cucurbitae plant which is widely produced in China, and a liquid extract (trade name: Xiaoaiping) thereof is approved to be used for treating esophageal cancer, lung cancer, leukemia, liver cancer and gastric cancer. It has been found that MTE is rich in C21 steroidal glycosides, and that treatment of FR5 fraction can induce apoptosis through PTEN-AKT-mTOR signaling pathway. However, the potential efficacy of C21 steroids on autophagy and whether inhibition of autophagy could promote the anticancer effects of C21 have not been investigated.

Therefore, the present invention mainly examines whether the C21 steroid can induce autophagy and whether autophagy inhibition contributes to the anticancer effect of the C21 extract, and also explores the potential molecular mechanism of inducing autophagy.

Disclosure of Invention

The invention aims to provide application of a C21 steroid in preparation of a medicine for enhancing cancer cell apoptosis, so as to solve the problems in the prior art, and the C21 steroid can obviously inhibit the growth of cancer cells and promote apoptosis so as to inhibit cell proliferation.

The invention also aims to provide application of the C21 steroid in preparation of a medicine for treating gastric cancer, wherein the C21 steroid has the effects of inducing apoptosis and autophagy increase of gastric cancer cells, and provides a theoretical basis for disclosing a new mechanism of anti-gastric cancer activity of the C21 steroid.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides an application of a C21 steroid in preparing a medicine for promoting cancer cell apoptosis, wherein the C21 steroid inhibits the proliferation of cancer cells by inhibiting autophagy to promote the apoptosis.

Preferably, the C21 steroid promotes the development of the autophagy pathway by activating AKT.

Preferably, the C21 steroid is glaucescent fissistigma root extract.

Preferably, the cancer cell is a gastric cancer cell.

The invention also provides application of the C21 steroid in preparation of a medicine for treating gastric cancer, wherein the C21 steroid is a glaucescent fissistigma root extract, and the C21 steroid inhibits proliferation of gastric cancer cells by inhibiting autophagy and promoting apoptosis.

The invention discloses the following technical effects:

since activation of the PI3K/Akt pathway has been demonstrated in many types of tumors, it contributes to the proliferation and survival of tumor cells. In the invention, experiments verify that the BGC-823 and AGS cells treated with C21 steroid have a significant reduction in p-AKT, indicating that C21 steroid may induce apoptosis through PTEN-AKT signaling pathway. And the inventor proves in previous research that the cochinchina sylvestris extract C21 steroid can induce the apoptosis of liver cancer cells through a PTEN/AKT/mTOR signaling pathway. The PI3K/Akt/mTOR pathway has been shown to play a negative role in regulating autophagy, its decline actually activates autophagy; it has also been found that an autophagy-related protein (ATG) plays a particular role in regulating the formation of autophagosomes encoded by autophagy-related genes. In the invention, the autophagy-related proteins ATG5 and Beclin-1 are increased when C21 steroid induces autophagy and are reduced when autophagy inhibition occurs, further indicating that the C21 steroid can activate autophagy in gastric cancer cells, and indicating that the C21 steroid can induce protective autophagy in gastric cancer cells by inhibiting PI3K/Akt/mTOR signaling pathway. The invention shows that the C21 steroid can inhibit gastric cancer cell proliferation, induce gastric cancer cell apoptosis and autophagy; in particular, C21 steroid enhances gastric cancer apoptosis by inhibiting autophagy, partly due to activation of AKT, and thus C21 steroid will be a new candidate for the treatment of gastric cancer, extracted from natural compounds.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows the effect of C21 steroid in inhibiting gastric cancer cell proliferation; a-C shows that C21 steroids at different concentrations inhibit BGC-823, SGC-7901 and AGS cell proliferation respectively: treating gastric cancer cells with a C21 steroid (0, 40, 80, 160. mu.g/mL, analyzing real-time cell detector results with Prism software from GraphPad. treating gastric cancer cells with a C21 steroid (0, 20, 40, 80, 160, 320. mu.g/mL), analyzing MTS results with Prism software from GraphPad. P <0.05, vs NC;

FIG. 2 is a graph showing induction of BGC823 and AGS cell cycle arrest and apoptosis in gastric cells treated with 0, 80 μ g/mL and 160 μ g/mL C21 steroids for 24 h; a: after staining with staining buffer a and reagent B, cell count (%) was determined by flow cytometry; b, cell cycle statistical chart; c: after being stained with Annexin-V-Fluorescein Isothiocyanate (FITC) and PI, the apoptosis rate was measured by flow cytometry; the lower left corner shows normal rate, the lower right corner early apoptosis, the upper right corner late apoptosis; d: a histogram of apoptosis rate; e: western blot analysis to determine clear-PARP protein levels, with β -actin as control; f: statistics of the expression rate of cleaned-PARP; p <0.05, vs NC;

figure 3 shows that C21 steroid treatment promotes gastric autophagy; a: co-localization of green fluorescent protein-LC 3 (green) was observed by confocal microscopy after 24h treatment with C21 steroid (100. mu.g/mL) in the absence or presence of CQ (20. mu.M); b: LC3 protein levels were determined by western blot analysis after exposing cells to C21 steroid (120. mu.g/mL) and CQ (20. mu.M) for 24h, with GAPDH as a control; c: quantifying expression rate statistics for LC 3-II; p <0.05, vs NC;

fig. 4 is the promotion of apoptosis by C21 steroid in combination with CQ; a: after being stained with Annexin-V-FITC and PI, the apoptosis rate is measured by a flow cytometer; the lower left corner is the normal ratio, the lower right corner is early apoptosis, the upper right corner is late apoptosis; b: quantifying the apoptosis percentage respectively; c: after DCFH-DA staining, quantitatively determining ROS level; a-b: after exposing the cells to C21 steroid (120. mu.g/mL) and CQ (20. mu.M) for 24h, the autophagy-related proteins were analyzed using western blot with GAPDH as control; c: quantifying the expression rate of Beclin-1; d: quantifying the expression rate of ATG-5; e: quantifying the expression rate of BCL-2; p <0.05, vs NC;

fig. 5 is a C21 steroid interfering with autophagy through the AKT signaling pathway; a-b: after BGC-823 and AGS cells were exposed to C21 steroid (120. mu.g/mL) and CQ (20. mu.M) for 24h, respectively, proteins associated with apoptosis were determined by Western blot analysis with GAPDH as a control; c: quantifying the expression rate of Beclin-1; d: quantifying the expression rate of ATG-5; e: quantifying the expression rate of p-AKT; p <0.05, vs NC.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Materials and methods

Fetal Bovine Serum (FBS) and Roswell Park clinical Institute 1640 Medium RPMI1640 was purchased from HyClone.

C21 steroids from glaucescent fissistigma root extract were provided by the institute of medicine laboratory of university of industry, zhejiang.

The GFP-LC3 plasmid was provided by the clinical laboratory of the people's hospital in Zhejiang province.

3- (45-dimethylthiozol-2-yl) -5- (3-carboxymethyxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium inner salt (MTS) was purchased from PROMEGA.

Chloroquine diphosphate was purchased from Sangon Biotech.

BGC-823, SGC-7901 and AGS cells-human gastric cancer cells-from the clinical laboratory in Min Hospital, Zhejiang province. All cells were cultured in Roswell Park clinical Institute medium 1640(RPMI1640, HyClone) supplemented with 10% fetal bovine serum (FBS, HyClone). All cells were cultured at 37 ℃ with 5% CO₂The incubator of (1).

Example 1

Inhibition of gastric cancer cell proliferation by C21 steroids

Add 50. mu.L of medium to each well of E-plate 96 (Roche) to obtain an equilibrium; 2000 cells were then seeded in E-plate 96. After 24 hours, 40. mu.g/mL, 80. mu.g/mL, 160. mu.g/mL C21 steroid were added, respectively, and the E-plate 96 was locked in an RTCA-MP apparatus at 37 ℃ with CO₂At a concentration of 5%, the measured changes in electrical impedance were recorded, which directly reflected the cell proliferation on the surface of the biocompatible microelectrode coating, and were read automatically every two minutes.

5000 cells were seeded in a 96-well plate containing 200. mu.L of RPIM1640 medium at 37 ℃ with 5% CO₂Cultured in the incubator of (1), then treated with C21 steroid (0, 20. mu.g/mL, 40. mu.g/mL, 80. mu.g/mL, 160. mu.g/mL, 320. mu.g/mL), cultured for 24 h; mu.L of MTS solution was added to each well, incubated for 2h, and absorbance was measured at 490nm using a microplate reader.

The results are shown in FIG. 1: the RTCA growth curves show that the C21 steroid greatly inhibited cell proliferation of BGC-823, SGC-7901, and AGS cells (fig. 1 a-C) in a concentration and time dependent manner. As shown in FIG. 1d, the MTS method detected IC in BGC-823, SGC-7901 and AGS cells₅₀. The results show that the C21 steroid remarkably inhibits the proliferation of gastric cancer cells and is dose-dependent. IC of BGC-823 cells₅₀IC of 80.57. mu.g/mL, SGC-7901 cells₅₀146.3 μ g/mL, IC of AGS cells₅₀Was 102.8. mu.g/mL.

The results show that C21 steroid stimulation has obvious inhibition effect on the proliferation of BGC-823, SGC-7901 and AGS cells.

Example 2

Cell cycle arrest and apoptosis inducing gastric cancer cells BGC-823 and AGS

Cells were seeded into 12-well plates incubated at 37 ℃ for 24h, then treated with C21 steroid (0, 80. mu.g/mL, 160. mu.g/mL) and incubated at 37 ℃ for 24h, cells were collected, treated with 500. mu.L staining buffer A and 5. mu.L reagent B (Union Biotechnology), and the cells were incubated in the dark at room temperature for 30min and analyzed for cell cycle by flow cytometry.

The percentage of apoptotic cells was determined by double staining with Annexin-V-FITC and PI (Biotech in Union) and then treated in the same manner as the cell cultures used in the cell cycle analysis. Cells were harvested, treated with 5. mu.L Annexin V-FITC and 10. mu.L PI, then cultured at room temperature in the dark for 5min, and analyzed for fluorescence-activated cells using flow cytometry.

The results are shown in FIGS. 2a and b: the cell cycle phase distribution of the treated cells is analyzed by flow cytometry, and the results show that 160 mu G/mL C21 steroids can increase the proportion of G2/M phase cells (16.68% + -0.93% and 15.37% + -0.48% of 160 mu G/mL C21 steroids respectively and 7.74% + -0.39% and 5.65% + -0.30% of control respectively) by reducing the percentage of G0/G1 phase cells (55.13% + -1.14% and 53.12% + -3.44% of 160 mu G/mL C21 steroids respectively and 66.33% + -1.37% and 66.83% + -2.26% of 160 mu G/mL C21 steroids respectively), and obviously inhibit the cell cycle progress of BGC823 and AGS cells.

As shown in fig. 2c and d, flow cytometry analysis determined whether cell growth inhibition was by apoptosis. Treatment of 24h gastric cancer cells with different doses of C21 steroid induced apoptosis, particularly late apoptosis, in a dose-dependent manner. Specifically, C21 steroid treatment increased the apoptosis rate of BGC-823 cells from 3.770% ± 0.393% to 5.133% ± 0.365% (80 μ g/mL) and 12.802% ± 2.090% (160 μ g/mL) of the control group; in AGS cells, the rate of apoptosis increased from 3.773% + -0.287% in the control group to 7.465% + -0.100% (80. mu.g/mL) and 13.075% + -0.928% (160. mu.g/mL).

As shown in fig. 2e and f, western blot analysis detected proteins associated with apoptosis. After 24h of C21 steroid treatment, both BGC-823 and AGS cell lines showed increased clear-PARP, suggesting that apoptosis may contribute to inhibition of cell growth by C21 steroids in gastric cancer cells.

Example 3

Reactive Oxygen Species (ROS) measurement

Cells were cultured for 24h in the presence or absence of C21 steroid alone or with the autophagy inhibitor CQ. After collection and resuspension in 500. mu.L PBS, cells were stained with DCFH-DA (5mm) for 30min according to the manufacturer's instructions (Beyotime), and then analyzed for DCFH-DA signals by flow cytometry.

To investigate the biological role of C21 steroid-induced autophagy in cell survival or death, gastric cancer cells were blocked from autophagy with the autophagy inhibitor CQ and treated with the C21 steroid alone or in combination with CQ for 24 h. As shown in fig. 4: the apoptosis experiment shows that CQ promotes the apoptosis of gastric cancer cells induced by C21 steroids. As shown in fig. 4a and 4b, the percentage of BGC-823 apoptosis increased by 11.20% ± 1.69% (C21 steroid) and 17.70% ± 0.39% (C21 steroid + CQ) compared to the control group. The percentage of AGS apoptosis increased by 8.79 ± 0.54% (C21 steroid) and 12.78% ± 1.23% (C21 steroid + CQ), respectively.

ROS production in gastric cancer cells was measured 24h after treatment with C21 steroid alone or in combination with CQ. As shown in fig. 4c, intracellular ROS production was increased during treatment compared to control cells. Compared with the control group, the ROS level of the BGC-823 cells is 1.512 +/-0.103 times (C21 steroid), 3.766 +/-0.724 times (CQ component) and 4.915 +/-0.077 times (C21 steroid + CQ group) of the control group respectively. The ROS level in AGS cells is 1.227 +/-0.088 times (C21 steroid), 1.698 +/-0.104 times (CQ component) and 2.848 +/-0.396 times (C21 steroid + CQ component) of the control group respectively.

Example 4

Confocal microscope for detecting LC3-II spots

Cells adhered to the slides were transfected with the GFP-LC3 plasmid using transfection reagents (YEASEN) according to the manufacturer's protocol. After 24h transfection, cells were treated with C21 steroid alone or CQ, incubated for 24h, stained with DAPI and an anti-quencher (1: 1000, Beyotime), and visualized with a confocal microscope (Leica).

The results show that: to assess the recruitment response of LC3-II to autophagosomes following C21 steroid treatment. As shown in figure 3a, a punctate LC3 pattern was observed in the cytoplasmic C21 steroid-treated cells, while untreated control cells showed diffuse and weak LC3 punctate spots.

The conversion of the lipidated form of LC3 from LC3-I to LC3-II is characteristic of autophagy. As shown in fig. 3b and 3C, LC3-II accumulated in gastric cancer cell lines after exposure to the C21 steroid; in addition, co-treatment with the autophagy inhibitor CQ blocked the last step of autophagy degradation and enhanced LC3-II accumulation induced by the C21 steroid.

Example 5

Western Blot analysis of autophagy flux proteins

Cells treated with C21 steroid were seeded into 12-well plates and cultured for 24 h. Total protein was extracted using a total cell protein extraction kit (RIPA, Beyotime). Equal amounts of protein from each sample were run on 12% SDS-PAGE and transferred to PVDF membrane. After blocking, the membrane was incubated with anti-PTEN (1: 1000; CST), anti-Bax (1: 1000; CST), anti-Bcl-2 (1: 1000; CST), anti-AKT (1: 1000; CST), anti-P-ARP (1: 1000; CST); anti-LC 3 (1: 1000; CST), anti-Beclin-1 (1: 1000; CST), overnight at 4 ℃ and then washed three times with PBS/0.1% Tween-20 for 10min each, then incubated with the corresponding secondary antibody (1: 2000, Waran biosciences) for 1h at room temperature and then washed three times again for 10min each. The membranes were detected using a chemiluminescent ECL kit (Fude biotechnology).

The level of apoptosis-related proteins in gastric cancer cells was detected using western blot analysis. As shown in fig. 4, C21 steroid treatment promoted protein expression of clear-PARP and Bax in both cell lines compared to the control; BCL-2 protein expression is obviously reduced. The level of autophagy-associated proteins in gastric cancer cells was detected by western blot analysis. As shown in FIG. 5, CQ reduced the C21 steroid-induced accumulation of Beclin-1 and A TG-5 proteins; in contrast, the expression of p-AKT was significantly reduced, a phenomenon that was consistent in both gastric cancer cells.

All of the assays of examples 1-5 above were performed in at least three separate experiments. The western blot analysis results were analyzed using image J software. All data were analyzed using the PRISM software from GraphPad, and all values are expressed as mean. + -. SD. Statistical differences were evaluated using independent t-tests and one-way analysis of variance. The significance of the difference was set at p < 0.05.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. The use of a C21 steroid for the preparation of a medicament for promoting apoptosis in cancer cells, wherein the C21 steroid inhibits proliferation of cancer cells by inhibiting autophagy to promote apoptosis.

2. The use of the C21 steroid for the preparation of a medicament for promoting apoptosis in cancer cells, wherein the C21 steroid promotes the development of the autophagy pathway by activating AKT.

3. The use of the C21 steroid of claim 1, wherein the C21 steroid is cochinchina sylvine extract for the preparation of a medicament for promoting apoptosis.

4. The use of the C21 steroid for the preparation of a medicament for promoting apoptosis in a human of claim 1, wherein the cancer cell is a gastric cancer cell.

5. The application of the C21 steroid in preparing a medicine for treating gastric cancer is characterized in that the C21 steroid is a glaucescent fissistigma root extract, and the C21 steroid inhibits proliferation of gastric cancer cells by inhibiting autophagy and promoting apoptosis.